Abstract:

The present invention encompasses arrays and methods related to the genome
of Methanobrevibacter-smithii.

Claims:

1. An array comprising a substrate, the substrate having disposed thereon
at least one nucleic acid, wherein the nucleic acid comprises a nucleic
acid sequence selected from the nucleic acid sequences listed in Table A.

2. The array of claim 1, wherein the nucleic acid or nucleic acids are
located at a spatially defined address of the array.

3. The array of claim 2, wherein the array has no more than 500 spatially
defined addresses.

4. The array of claim 2, wherein the array has at least 500 spatially
defined addresses.

5. A method of selecting a compound that has efficacy for modulating a
gene product of M. smithii present in the gastrointestinal tract of a
subject, the method comprising:a. comparing an M. smithii gene profile to
a gene profile of the subject,b. identifying a gene product of the M.
smithii gene profile that is divergent from a corresponding gene product
of the subject gene profile, or absent in the gene profile of the
subject, andc. selecting a compound that modulates the M. smithii gene
product but does not substantially modulate the corresponding divergent
gene product of the subject.

6. The method of claim 5, wherein the compound inhibits the M. smithii
gene product, but does not substantially inhibit the corresponding gene
product of the subject.

7. The method of claim 6, wherein the compound inhibits the growth of M.
smithii.

8. The method of claim 6, wherein the compound decreases the efficiency of
carbohydrate metabolism in the subject.

9. The method of claim 6, wherein the compound promotes weight loss.

10. The method of claim 5, wherein the compound upregulates the M. smithii
gene product, but does not substantially upregulate the corresponding
gene product of the subject.

11. The method of claim 10, wherein the compound promotes the growth of M.
smithii.

12. The method of claim 10, wherein the compound increases the efficiency
of carbohydrate metabolism in the subject.

14. The method of claim 5, wherein the compound, as administered to a
subject, modulates the M. smithii gene product but does not substantially
modulate the corresponding divergent gene product of the subject.

15. The method of claim 14, wherein the compound is an HMG-CoA reductase
inhibitor.

16. A method for modulating a gene product of M. smithii present in the
gastrointestinal tract of a subject, the method comprising administering
to the subject an HMG-CoA reductase inhibitor that has been formulated
for release in the distal portion of the subject's gastrointestinal tract
and thereby substantially inhibits more of the HMG-CoA reductase of M.
smithii compared to the subject's HMG-CoA reductase.

17. The method of claim 16, wherein the inhibitor is a statin.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the priority of PCT application
PCT/US2008/065344, filed May 30, 2008, which claims the priority of U.S.
provisional application No. 60/932,457, filed May 31, 2007, each of which
is hereby incorporated by reference in its entirety.

[0004]According to the Center for Disease Control (CDC), over sixty
percent of the United States population is overweight, and almost twenty
percent are obese. This translates into 38.8 million adults in the United
States with a Body Mass Index (BMI) of 30 or above. Obesity is also a
world-wide health problem with an estimated 500 million overweight adult
humans [body mass index (BMI) of 25.0-29.9 kg/m2] and 250 million
obese adults. This epidemic of obesity is leading to worldwide increases
in the prevalence of obesity-related disorders, such as diabetes,
hypertension, as well as cardiac pathology, and non-alcoholic fatty liver
disease (NAFLD).

[0005]According to the National Institute of Diabetes, Digestive and
Kidney Diseases (NIDDK) approximately 280,000 deaths annually are
directly related to obesity. The NIDDK further estimated that the direct
cost of healthcare in the U.S. associated with obesity is $51 billion. In
addition, Americans spend $33 billion per year on weight loss products.
In spite of this economic cost and consumer commitment, the prevalence of
obesity continues to rise at alarming rates. From 1991 to 2000, obesity
in the U.S. grew by 61%.

[0006]Additionally, malnourishment or disease may lead to individuals
being under weight. The World Health Organization estimates that
one-third of the world is under-fed and one-third is starving. Over 4
million will die this year from malnourishment. One in twelve people
worldwide is malnourished, including 160 million children under the age
of 5.

II. Gastrointestinal Microbiota

[0007]Humans are host to a diverse and dynamic population of microbial
symbionts, with the majority residing within the distal intestine. The
gut microbiota contains representatives from ten known divisions of the
domain Bacteria, with an estimated 500-1000 species-level phylogenetic
types present in a given healthy adult human; the microbiota is dominated
by members of two divisions of Bacteria, the Bacteroidetes and the
Firmicutes. Members of the domain Archaea are also represented, most
prominently by a methanogenic Euryarchaeote, Methanobrevibacter smithii
and occasionally Methanosphaera stadtmanae. The density of colonization
increases by eight orders of magnitude from the proximal small intestine
(103) to the colon (1011). The distal intestine is an anoxic
bioreactor whose microbial constituents help the subject by providing a
number of key functions: e.g., breakdown of otherwise indigestible plant
polysaccharides and regulating subject storage of the extracted energy;
biotransformation of conjugated bile acids and xenobiotics; degradation
of dietary oxalates; synthesis of essential vitamins; and education of
the immune system.

[0008]Dietary fiber is a key source of nutrients for the microbiota.
Monosaccharides are absorbed in the proximal intestine, leaving dietary
fiber that has escaped digestion (e.g. resistant starches, fructans,
cellulose, hemicelluloses, pectins) as the primary carbon sources for
microbial members of the distal gut. Fermentation of these
polysaccharides yields short-chain fatty acids (SCFAs; mainly acetate,
butyrate and propionate) and gases (H2 and CO2). These end
products benefit humans. For example, SCFAs are an important source of
energy, as they are readily absorbed from the gut lumen and are
subsequently metabolized in the colonic mucosa, liver, and a variety of
peripheral tissues (e.g., muscle). SCFAs also stimulate colonic blood
flow and the uptake of electrolytes and water.

III. Methanogens

[0009]Methanogens are members of the domain Archaea. Methanogens thrive in
many anaerobic environments together with fermentative bacteria. These
habitats include natural wetlands as well as man-made environments, such
as sewage digesters, landfills, and bioreactors. Hydrogen-consuming,
mesophilic methanogens are also present in the intestinal tracts of many
invertebrate and vertebrate species, including termites, birds, cows, and
humans. Using methane breath tests, clinical studies estimate that
between 30 and 80 percent of humans harbor methanogens.

[0010]Culture- and non-culture-based enumeration studies have demonstrated
that members of the Methanobrevibacter genus are prominent gut mesophilic
methanogens. The most comprehensive enumeration of the adult human
colonic microbiota reported to date found a single predominant archaeal
species, Methanobrevibacter smithii. This gram-positive-staining
Euryarchaeote can comprise up to 1010 cells/g feces in healthy
humans, or ˜10% of all anaerobes in the colons of healthy adults.

[0011]A focused set of nutrients are consumed for energy by methanogens:
primarily H2/CO2, formate, acetate, but also methanol, ethanol,
methylated sulfur compounds, methylated amines and pyruvate. These
compounds are typically converted to CO2 and methane (e.g. acetate)
or reduced with H2 to methane alone (e.g. methanol or CO2).
Some methanogens are restricted to utilizing only H2/CO2 (e.g.
Methanobrevibacter arbophilicus), or methanol (e.g. Methanospaera
stadtmanae). Other more ubiquitous methanogens exhibit greater metabolic
diversity, like Methanosarcina species. In vitro studies suggest that M.
smithii is intermediate in this metabolic spectrum, consuming
H2/CO2 and formate as energy sources.

IV. Anaerobic Microbial Fermentation in the Mammalian Intestine

[0012]Fermentation of dietary fiber is accomplished by syntrophic
interactions between microbes linked in a metabolic food web, and is a
major energy-producing pathway for members of the Bacteroidetes and the
Firmicutes. Bacteroides thetaiotaomicron has previously been used as a
model bacterial symbiont for a variety of reasons: (i) it effectively
ferments a range of otherwise indigestible plant polysaccharides in the
human colon; (ii) it is genetically manipulatable; and, (iii) it is a
predominant member of the human distal intestinal microbiota. Its 6.26 Mb
genome has been sequenced: the results reveal that B. thetaiotaomicron
has a large collection of known or predicted glycoside hydrolases (261 in
total; by comparison, our human genome only encodes 99 known or predicted
glycoside hydrolases). B. thetaiotaomicron also has a significant
expansion of outer membrane polysaccharide binding and importing proteins
(over 208 paralogs of two starch binding proteins known as SusC and
SusD), as well as a large repertoire of environmental sensing proteins
[e.g. 50 extra-cytoplasmic function (ECF)-type sigma factors; 25
anti-sigma factors, and 32 novel hybrid two-component systems].
Functional genomics studies of B. thetaiotaomicron in vitro and in the
ceca of gnotobiotic mice, indicates that it is capable of very flexible
foraging for dietary (and host-derived) polysaccharides, allowing this
organism to have a broad niche and contributing to the functional
stability of the microbiota in the face of changes in the diet.

[0013]In vitro biochemical studies of B. thetaiotaomicron and closely
related Bacteroides species (B. fragilis and B. succinogenes) indicate
that their major end products of fermentation are acetate, succinate,
H2 and CO2. Small amounts of pyruvate, formate, lactate and
propionate are also formed.

V. Removal of Hydrogen from the Intestinal Ecosystem is Important for
Efficient Microbial Fermentation

[0014]Anaerobic fermentation of sugars causes flux through glycolytic
pathways, leading to accumulation of NADH (via glyceraldehyde-3P
dehydrogenase) and the reduced form of ferredoxin (via
pyruvate:ferredoxin oxidoreductase). B. thetaiotaomicron is able to
couple NAD.sup.+ recovery to reduction of pyruvate to succinate (via
malate dehydrogenase and fumarase reductase), or lactate (via lactate
dehydrogenase). Oxidation of reduced ferredoxin is easily coupled to
production of H2. However, H2 formation is, in principle, not
energetically feasible at high partial pressures of the gas. In other
words, lower partial pressures of H2 (1-10 Pa) allow for more
complete oxidation of carbohydrate substrates. The subject removes some
hydrogen from the colon by excretion of the gas in the breath and as
flatus. However, the primary mechanism for eliminating hydrogen is by
interspecies transfer from bacteria by hydrogenotrophic methanogens.
Formate and acetate can also be transferred between some species, but
their transfer is complicated by their limited diffusion across the
lipophilic membranes of the producer and consumer. In areas of high
microbial density or aggregation like in the gut, interspecies transfer
of hydrogen, formate and acetate is likely to increase with decreasing
physical distance between microbes.

[0015]Methanogen-mediated removal of hydrogen can have a profound impact
on bacterial metabolism. Not only does re-oxidation of NADH occur, but
end products of fermentation undergo a shift from a mixture of acetate,
formate, H2, CO2, succinate and other organic acids to
predominantly acetate and methane with small amounts of succinate. This
facilitates disposal of reducing equivalents, and produces a potential
gain in ATP production due to increased acetate levels. For example, a
reduction in hydrogen allows Clostridium butyricum to acquire 0.7 more
ATP equivalents from fermentation of hexose sugars. Co-culture of M.
smithii with a prominent cellulolytic ruminal bacterial species,
Fibrobacter succinogenes S85, results in augmented fermentation, as
manifested by increases in the rate of ATP production and organic acid
concentrations. Co-culture of M. smithii association with Ruminococcus
albus eliminates NADH-dependent ethanol production from acetyl-CoA,
thereby skewing bacterial metabolism towards production of acetate, which
is more energy yielding. H2-producing fibrolytic bacterial strains
from the human colon exhibit distinct cellulose degradation phenotypes
when co-cultured with M. smithii, indicating that some bacteria are more
responsive to syntrophy with methanogens.

[0016]While there is suggestive evidence that methanogens cooperate
metabolically with members of Bacteroides, studies have not elucidated
the impact of this relationship on a subject's energy storage or on the
specificity and efficiency of carbohydrate metabolism. Colonization of
adult germ-free mice with M. smithii and/or B. thetaiotaomicron, revealed
that the methanogen increased the efficiency and changed the specificity
of bacterial digestion of dietary glycans. Moreover, co-colonized mice
exhibited a significantly greater increase in adiposity compared with
mice colonized with either organism alone.

SUMMARY OF THE INVENTION

[0017]One aspect of the present invention encompasses an array. The array
comprises a substrate having disposed thereon at least one nucleic add,
wherein the nucleic acid comprises a nucleic acid sequence selected from
the nucleic acid sequences listed in Table A.

[0018]Another aspect of the present invention encompasses an array. The
array comprises a substrate having disposed thereon at least one
polypeptide, wherein the polypeptide is encoded by a nucleic acid
sequence selected from the nucleic acid sequences listed in Table A.

[0019]Yet another aspect of the present invention encompasses a method of
selecting a compound that has efficacy for modulating a gene product of
M. smithii present in the gastrointestinal tract of a subject. The method
comprises comparing an M. smithii gene profile to a gene profile of the
subject, identifying a gene product of the M. smithii gene profile that
is divergent from a corresponding gene product of the subject gene
profile, or absent in the gene profile of the subject, and selecting a
compound that modulates the M. smithii gene product but does not
substantially modulate the corresponding divergent gene product of the
subject.

[0020]Still another aspect of the invention encompasses a method for
modulating a gene product of M. smithii present in the gastrointestinal
tract of a subject. The method comprises administering to the subject an
HMG-CoA reductase inhibitor. The inhibitor may be formulated for release
in the distal portion of the subject's gastrointestinal tract and thereby
substantial inhibit more of the HMG-CoA reductase of M. smithii compared
to the subject's HMG-CoA reductase.

[0021]Other aspects and iterations of the invention are described more
thoroughly below.

REFERENCE TO COLOR FIGURES

[0022]The application file contains at least one photograph executed in
color. Copies of this patent application publication with color
photographs will be provided by the Office upon request and payment of
the necessary fee.

[0025]FIG. 3. depicts a diagram of the analysis of the M. smithii
pan-genome. Schematic depiction of the conservation of M. smithii PS
genes [depicted in the outermost circle where the color code is orange
for forward strand ORFs (F) and blue for reverse strand ORFs (R)] in (i)
other M. smithii strains (GeneChip-based genotyping of strains F1, ALI,
and B181; circles in increasingly lighter shades of green, respectively),
(ii) the fecal microbiomes of two healthy individuals [human gut
microbiome (HGM), shown as the red plot in the fifth innermost circle
with nucleotide identity plotted from 80% (closest to the purple circle)
to 100% (closest to lightest green ring); see also FIG. 9 for details],
and (iii) two other members of the Methanobacteriales division, M.
stadtmanae (Msp; purple circle), another human gut methanogen, and M.
thermoautotrophicus (Mth; yellow circle), an environmental thermophile
[mutual best blastp hits (e-value <10-20)]. Tick marks in the
center of the Figure indicate nucleotide number in kbps. Asterisks denote
the positions of ribosomal rRNA operons. Letters highlight distinguishing
features among M. smithii genomes: the table below the figure summarizes
differences in M. smithii gene content between strains F1, ALI, and B181
as well as the two human fecal metagenomic datasets.

[0030]FIG. 8. depicts an illustration showing the importance of the
molybdopterin biosynthesis pathway for methanogenesis from carbon dioxide
in M. smithii. (A) In silico metabolic reconstruction of the predicted
molybdopterin biosynthesis pathway encoded by the M. smithii genome.
Molybdopterin can chelate molybdate (MoO4.sup.-) or tungstate
(WO42-) ions. Abbreviations: MoaABCE, molybdenum cofactor
biosynthesis proteins A (MSM0849, MSM1406), B (MSM0840), C (MSM1362), and
E (MSM0130); MoeAB, molybdopterin biosynthesis proteins A (MSM1343) and B
(MSM0729); ModABC, molybdate ABC transport system (MSM1609-11); MobAB,
molybdopterin-guanine dinucleotide (MGD) biosynthesis proteins A
(MSM0240) and B (MSM1407); PP, pyrophosphate. Note that the molybdate
transporter may also be used for WO42-, as no dedicated complex
has been identified for its transport. (B) Schematic of the first step in
the methanogenesis pathway from carbon dioxide (CO2) catalyzed by
tungsten-containing formylmethanofuran dehydrogenase (Fwd; MSM1408-14,
MSM0783, MSM1396). Essential cofactors for this reaction include tungsten
delivered by MGD, methanofuran (MFN), and ferridoxin [Fd; converted from
a reduced (red) to oxidized (ox) form during the reaction].

[0031]FIG. 9. illustrates the divergence in genes involved in surface
variation, genome evolution, and metabolism among M. smithii strains and
in the human gut microbiomes of two healthy adults. Each of the 139,521
unidirectional reads in the metagenomic dataset (Gill et al., (2006)
Science 312, 1355-9) were compared to the M. smithii PS genome using
NUCmer. Reads with nucleotide sequence identity ≧80% (present) are
plotted. A summary of representation of M. smithii PS genes present in
the metagenomic dataset is displayed at the bottom of the graph (92% of
the total ORFs). [Note that the gaps are indications of genome plasticity
in the dataset, and include transposases, restriction-modification
systems and prophage genes.] Selected regions of heterogeneity
(divergence) are highlighted; genes in these regions are involved in the
metabolism of bacterial products, recombination/repair machinery
(Recomb), anti-microbial resistance (AntiMicrob), surface variation
(Surface), and adhesion (ALPs). See Table 2 for details.

[0037]FIG. 15 depicts two photographs of the PHAT system described in the
Examples. Panel A shows the pressurized incubation vessels within the
anaerobic chamber, while Panel B shows an individual PHAT system outside
of the chamber.

DETAILED DESCRIPTION

[0038]The present invention provides arrays and methods utilizing the
genome and proteome of the methanogen M. smithii, which is the
predominant methanogen present in the human gastrointestinal tract.
Modulating the Archaeal population of the gastrointestinal tract of a
subject, of which M. smithii is a major component, modulates the
efficiency and selectivity of carbohydrate metabolism. The genome and
proteome of M. smithii may be used, according to the methods presented
herein, to promote weight loss or weight gain in a subject. In
particular, the methods of the present invention may be used to identify
compounds that promote weight loss or weight gain in a subject. The
method relies on applicants' discovery that certain M. smithii gene
products are conserved between M. smithii strains, yet divergent (or
absent) from the correlating gene products expressed by the subject's
microbiome or genome. This allows the selection of compounds that
specifically modulate the M. smithii gene product, while substantially
not modulating the subject's gene product.

I. Arrays

[0039]One aspect of the invention encompasses use of biomolecules in an
array. As used herein, biomolecule refers to either nucleic acids derived
from the M. smithii genome, or polypeptides derived from the M. smithii
proteome. The M. smithii genome or proteome may be utilized to construct
arrays that may be used for several applications, including discovery of
compounds that modulate one or more M. smithii gene products, judging
efficacy of existing weight gain or loss regimes, and for the
identification of biomarkers involved in weight gain or loss, or a weight
gain or loss related disorder.

[0040]The array may be comprised of a substrate having disposed thereon at
least one biomolecule. Several substrates suitable for the construction
of arrays are known in the art. The substrate may be a material that may
be modified to contain discrete individual sites appropriate for the
attachment or association of the biomolecule and is amenable to at least
one detection method. Alternatively, the substrate may be a material that
may be modified for the bulk attachment or association of the biomolecule
and is amenable to at least one detection method. Non-limiting examples
of substrate materials include glass, modified or functionalized glass,
plastics (including acrylics, polystyrene and copolymers of styrene and
other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, TeflonJ, etc.), nylon or nitrocellulose, polysaccharides,
nylon, resins, silica or silica-based materials including silicon and
modified silicon, carbon, metals, inorganic glasses and plastics. In an
exemplary embodiment, the substrates may allow optical detection without
appreciably fluorescing.

[0041]A substrate may be planar, a substrate may be a well, i.e. a 1534-,
384-, or 96-well plate, or alternatively, a substrate may be a bead.
Additionally, the substrate may be the inner surface of a tube for
flow-through sample analysis to minimize sample volume. Similarly, the
substrate may be flexible, such as a flexible foam, including closed cell
foams made of particular plastics. Other suitable substrates are known in
the art.

[0042]The biomolecule or biomolecules may be attached to the substrate in
a wide variety of ways, as will be appreciated by those in the art. The
biomolecule may either be synthesized first, with subsequent attachment
to the substrate, or may be directly synthesized on the substrate. The
substrate and the biomolecule may both be derivatized with chemical
functional groups for subsequent attachment of the two. For example, the
substrate may be derivatized with a chemical functional group including,
but not limited to, amino groups, carboxyl groups, oxo groups or thiol
groups. Using these functional groups, the biomolecule may be attached
using functional groups on the biomolecule either directly or indirectly
using linkers.

[0043]The biomolecule may also be attached to the substrate
non-covalently. For example, a biotinylated biomolecule can be prepared,
which may bind to surfaces covalently coated with streptavidin, resulting
in attachment. Alternatively, a biomolecule or biomolecules may be
synthesized on the surface using techniques such as photopolymerization
and photolithography. Additional methods of attaching biomolecules to
arrays and methods of synthesizing biomolecules on substrates are well
known in the art, i.e. VLSIPS technology from Affymetrix (e.g., see U.S.
Pat. No. 6,566,495, and Rockett and Dix, Xenobiotica 30(2):155-177, each
of which is hereby incorporated by reference in its entirety).

[0044]In one embodiment, the biomolecule or biomolecules attached to the
substrate are located at a spatially defined address of the array. Arrays
may comprise from about 1 to about several hundred thousand addresses. In
one embodiment, the array may be comprised of less than 10,000 addresses.
In another alternative embodiment, the array may be comprised of at least
10,000 addresses. In yet another alternative embodiment, the array may be
comprised of less than 5,000 addresses. In still another alternative
embodiment, the array may be comprised of at least 5,000 addresses. In a
further embodiment, the array may be comprised of less than 500
addresses. In yet a further embodiment, the array may be comprised of at
least 500 addresses.

[0045]A biomolecule may be represented more than once on a given array. In
other words, more than one address of an array may be comprised of the
same biomolecule. In some embodiments, two, three, or more than three
addresses of the array may be comprised of the same biomolecule. In
certain embodiments, the array may comprise control biomolecules and/or
control addresses. The controls may be internal controls, positive
controls, negative controls, or background controls.

[0046]The biomolecule may be a nucleic acid derived from the M. smithii
genome (GenBank Accession number CP000678), comprising, in part, nucleic
acid sequences labeled MSM001 through MSM1795, inclusive. Such nucleic
acids may include RNA (including mRNA, tRNA, and rRNA), DNA, and
naturally occurring or synthetically created derivatives. A nucleic acid
derived from the M. smithii genome is a nucleic acid that comprises at
least a portion of a nucleic acid sequence selected from the nucleic acid
sequences listed in Table A. The nucleic acid may comprise fewer than 10,
at least 10, at least 20, at least 30, at least 40, at least 50, at least
60, at least 70, at least 80, at least 90, at least 100, at least 150, at
least 200, or more than 200 bases of a nucleic acid sequence selected
from the nucleic acid sequences listed in Table A. One embodiment of the
invention is an array comprising a substrate, the substrate having
disposed thereon at least one nucleic acid, wherein the nucleic acid
comprises a nucleic acid sequence selected from the nucleic acid
sequences listed in Table A. In another embodiment, the nucleic acid
consists of a nucleic acid sequence selected from the nucleic acid
sequences listed in Table A. In certain embodiments, the nucleic acid
comprises a nucleic acid sequence derived from a sequence in Table A
marked by an asterick. The asterick marks sequences associated with a
core gut-associated M. smithii genome.

[0047]In one embodiment, the nucleic acid or nucleic acids may be selected
from the group of nucleic acids listed in Table A that are conserved
among M. smithii strains, but divergent from a corresponding nucleic acid
of the subject. In this context, a "corresponding nucleic acid" refers to
a nucleic acid sequence of the subject, or the subject's micobiome, that
has greater than 75% identity to a nucleic acid sequence of Table A. The
term, "divergent," as used herein, refers to a sequence of Table A that
has less than 99% identity, but greater than 75% identity, with a nucleic
acid sequence of the subject, or the subject's microbiome. For instance,
in some embodiments, divergent refers to less than or equal to about 98%,
97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%,
83%, 82%, 81%, 80%, 79%, 78%, 77%, or 76%, identity between the nucleic
acid sequence of Table A and the nucleic acid sequence of the subject.
Conversely, the term "conserved," as used herein, refers to a nucleic
acid sequence of one M. smithii strain that has greater than about 90%
identity to a nucleic acid sequence from another M. smithii strain.

[0048]If a subject, or the subject's microbiome, does not comprise a
nucleic acid sequence that has greater than 75% identity to a nucleic
acid sequence of Table A, that nucleic acid sequence of Table A is
"absent" from the subject. In certain embodiments, the nucleic acid or
nucleic acids of the array of the invention are selected from the group
comprising nucleic acid sequences that are absent from the subject gut
microbiome or genome. For instance, in one embodiment, the nucleic acid
may be selected from the group of nucleic acids designated absent or
divergent in Table 2. Percent identity may be determined as discussed
below.

[0049]Alternatively, the nucleic acid or nucleic acids derived from the M.
smithii genome (Table A) may be selected from the group of nucleic acids
comprising nucleic acid sequences that are expressed in vivo by M.
smithii while residing in the gastrointestinal tract of a subject. In
another embodiment, the nucleic acid or nucleic acids may be selected
from the group of nucleic acids comprising nucleic acid sequences that
are expressed by M. smithii while residing in the gastrointestinal tract
of a subject, and whose expression levels are not affected by the
presence of actively fermenting bacteria. In another embodiment, the
nucleic acid or nucleic acids may be selected from the group of nucleic
acids comprising nucleic acid sequences that are expressed by M. smithii
while residing in the gastrointestinal tract of a subject, and whose
expression levels are affected by the presence of actively fermenting
bacteria. The in vivo expression levels of a nucleic acid may be
determined by methods known in the art, including RT-PCR. In yet another
embodiment, the nucleic acid or nucleic acids may be selected from the
group of nucleic acids that encode the M. smithii transcriptome or
metabolome.

[0050]The biomolecule may also be a polypeptide derived from the M.
smithii proteome. A polypeptide derived from the M. smithii proteome is a
polypeptide that is encoded by at least a portion of a nucleic acid
sequence selected from the nucleic acid sequences listed in Table A. The
polypeptide may comprise fewer than 10, at least 10, at least 20, at
least 30, at least 40, at least 50, at least 60, at least 70, at least
80, at least 90, at least 100, at least 150, at least 200, or more than
200 amino acids encoded by a nucleic acid sequence selected from the
nucleic acid sequences listed in Table A. One embodiment of the invention
is an array comprising a substrate, the substrate having disposed thereon
at least one polypeptide, wherein the polypeptide is encoded by a nucleic
acid sequence selected from the nucleic acid sequences listed in Table A.
In certain embodiments, a biomolecule may be an amino acid sequence
derived from a sequence in Table A marked by an asterick. The asterick
marks sequences associated with a core gut-associated M. smithii genome.

[0051]In one embodiment, the polypeptide or polypeptides may be selected
from the group of polypeptides comprising polypeptide sequences that are
conserved among M. smithii strains, but divergent from a corresponding
polypeptide of the subject. The terms conserved and divergent are used as
defined above. In certain embodiments, the polypeptide or polypeptides
are selected from the group comprising polypeptides absent from the
subject gut microbiome or genome. In another embodiment, the polypeptide
or polypeptides may be selected from the group of polypeptides comprising
polypeptide sequences with greater than about 75% but less than about 99%
identity to a correlating polypeptide from the subject gut microbiome or
genome. In yet another embodiment, the polypeptide or polypeptides may be
selected from the group of polypeptides comprising polypeptide sequence
with greater than about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%
identity to a correlating polypeptide from the subject gut microbiome or
genome. In one embodiment, for instance, the polypeptide may be encoded
by a nucleic acid designated absent or divergent in Table 2. Percent
identity may be determined as discussed below.

[0052]Alternatively, the polypeptide or polypeptides derived from the M.
smithii proteome (see Table A) may be encoded by a nucleic acid selected
from the group of nucleic acids comprising nucleic acid sequences that
are expressed in vivo by M. smithii while residing in the
gastrointestinal tract of a subject. In another embodiment, the
polypeptide or polypeptides may be encoded by a nucleic acid selected
from the group of nucleic acids comprising nucleic acid sequences that
are expressed by M. smithii while residing in the gastrointestinal tract
of a subject, and whose expression levels are not affected by the
presence of actively fermenting bacteria. In still another embodiment,
the polypeptide or polypeptides may be encoded by a nucleic acid selected
from the group of nucleic acids comprising nucleic acid sequences that
are expressed by M. smithii while residing in the gastrointestinal tract
of a subject, and whose expression levels are affected by the presence of
actively fermenting bacteria. In yet another embodiment, the polypeptide
or polypeptides may be encoded by a nucleic acid selected from the group
of nucleic acids that encode the M. smithii transcriptome or metabolome.

[0053]The array may alternatively be comprised of biomolecules from the
genome or proteome of M. smithii that are indicative of an obese subject
microbiome. Alternatively, the array may be comprised of biomolecules
from the genome or proteome of M. smithii that are indicative of a lean
subject microbiome. A biomolecule is "indicative" of an obese or lean
microbiome if it tends to appear more often in one type of microbiome
compared to the other. Such differences may be quantified using commonly
known statistical measures, such as binomial tests. An "indicative"
biomolecule may be referred to as a "biomarker."

[0054]Additionally, the array may be comprised of biomolecules from the
genome or proteome of M. smithii that are modulated in the obese subject
microbiome compared to the lean subject microbiome. As used herein,
"modulated" may refer to a biomolecule whose representation or activity
is different in an obese subject microbiome compared to a lean subject
microbiome. For instance, modulated may refer to a biomolecule that is
enriched, depleted, up-regulated, down-regulated, degraded, or stabilized
in the obese subject microbiome compared to a lean subject microbiome. In
one embodiment, the array may be comprised of a biomolecule enriched in
the obese subject microbiome compared to the lean subject microbiome. In
another embodiment, the array may be comprised of a biomolecule depleted
in the obese subject microbiome compared to the lean subject microbiome.
In yet another embodiment, the array may be comprised of a biomolecule
up-regulated in the obese subject microbiome compared to the lean subject
microbiome. In still another embodiment, the array may be comprised of a
biomolecule down-regulated in the obese subject microbiome compared to
the lean subject microbiome. In still yet another embodiment, the array
may be comprised of a biomolecule degraded in the obese subject
microbiome compared to the lean subject microbiome. In an alternative
embodiment, the array may be comprised of a biomolecule stabilized in the
obese subject microbiome compared to the lean subject microbiome.

[0055]Additionally, the biomolecule may be at least 80, 85, 90, or 95%
homologous to a biomolecule derived from Table A. In one embodiment, the
biomolecule may be at least 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89%
homologous to a biomolecule derived from Table A. In another embodiment,
the biomolecule may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% homologous to a biomolecule derived from Table A.

[0057]In determining whether a biomolecule is substantially homologous or
shares a certain percentage of sequence identity with a sequence of the
invention, sequence similarity may be determined by conventional
algorithms, which typically allow introduction of a small number of gaps
in order to achieve the best fit. In particular, "percent identity" of
two polypeptides or two nucleic acid sequences is determined using the
algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA
87:2264-2268, 1993). Such an algorithm is incorporated into the BLASTN
and BLASTX programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990).
BLAST nucleotide searches may be performed with the BLASTN program to
obtain nucleotide sequences homologous to a nucleic acid molecule of the
invention. Equally, BLAST protein searches may be performed with the
BLASTX program to obtain amino acid sequences that are homologous to a
polypeptide of the invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST is utilized as described in Altschul et al.
(Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped
BLAST programs, the default parameters of the respective programs (e.g.,
BLASTX and BLASTN) are employed. See http://www.ncbi.nlm.nih.gov for more
details.

[0058]Furthermore, the biomolecules used for the array may be labeled. One
skilled in the art understands that the type of label selected depends in
part on how the array is being used. Suitable labels may include
fluorescent labels, chromagraphic labels, chemi-luminescent labels, FRET
labels, etc. Such labels are well known in the art.

II. Use of the Arrays

[0059]The arrays may be utilized in several suitable applications. For
example, the arrays may be used in methods for detecting association
between a biomolecule of the array and a compound in a sample. In this
context, compound refers to a nucleic acid, a protein, a lipid, or
chemical compound. In some embodiments, a compound may be an antibody.
This method typically comprises incubating a sample with the array under
conditions such that the compounds comprising the sample may associate
with the biomolecules attached to the array. The association is then
detected, using means commonly known in the art, such as fluorescence.
"Association," as used in this context, may refer to hybridization,
covalent binding, ionic binding, hydrogen binding, van der Waals binding,
and dated binding. A skilled artisan will appreciate that conditions
under which association may occur will vary depending on the
biomolecules, the compounds, the substrate, and the detection method
utilized. As such, suitable conditions may have to be optimized for each
individual array created.

[0060]In one embodiment, the array may be used as a tool in methods to
determine whether a compound has efficacy for modulating a gene product
of M. smithii. In certain embodiments, the array may be used as a tool in
methods to determine whether a compound has efficacy for modulating a
gene product of M. smithii while M. smithii is residing in the
gastrointestinal tract of a subject. Typically, such a method comprises
comparing a plurality of biomolecules from either the M. smithii genome
or proteome before and after administration of a compound for modulating
a gene product of M. smithii, such that if the abundance of a biomolecule
that correlates with the gene product is modulated, the compound is
efficacious in modulating a gene product of M. smithii. The array may
also be used to quantitate the plurality of biomolecule's of M. smithii's
genome or proteome before and after administration of a compound. The
abundance of each biomolecule in the plurality may then be compared to
determine if there is a decrease in the abundance of biomolecules
associated with the compound. In other embodiments, the array may be used
to quantify the levels of M. smithii in an obese subject prior to,
during, or after treatment for obesity. Alternatively, the array may be
used to quantify the levels of M. smithii in an underfed individual prior
to, during, or after implementation of dietary recommendations designed
to increase nutrient and energy harvest.

[0061]In a further embodiment, the array may be used as a tool in methods
to determine whether a compound has efficacy for treatment of weight gain
or a weight gain related disorder in a subject. Typically, such a method
comprises comparing a plurality of biomolecules of M. smithii's genome or
proteome before and after administration of a compound for the treatment
of weight gain or a weight gain related disorder, such that if the
abundance of biomolecules associated with weight gain decreased after
treatment, the compound is efficacious in treating weight gain in a
subject.

[0062]In still a further embodiment, the array may be used as a tool in
methods to determine whether a compound has efficacy for treatment of
weight loss or a weight loss related disorder in a subject. Typically,
such a method comprises comparing a plurality of biomolecules of M.
smithii's genome or proteome before and after administration of a
compound for the treatment of weight loss or a weight loss related
disorder, such that if the abundance of biomolecules associated with
weight loss decreased after treatment, the compound is efficacious in
treating weight loss in a subject.

[0063]In an alternative embodiment, a proteome array of the invention may
be used to screen antibodies that bind to one or more sequences of the M.
smithii proteome.

[0064]The present invention also encompasses M. smithii gene profiles.
Generally speaking, a gene profile is comprised of a plurality of values
with each value representing the abundance of a biomolecule derived from
either the M. smithii genome or proteome. The abundance of a biomolecule
may be determined, for instance, by sequencing the nucleic acids of the
M. smithii genome as detailed in the examples. This sequencing data may
then be analyzed by known software to determine the abundance of a
biomolecule in the analyzed sample. An M. smithii gene profile may
comprise biomolecules from more than one M. smithii strain. The abundance
of a biomolecule may also be determined using an array described above.
For instance, by detecting the association between compounds comprising
an M. smithii derived sample and the biomolecules comprising the array,
the abundance of M. smithii biomolecules in the sample may be determined.

[0065]A profile may be digitally-encoded on a computer-readable medium.
The term "computer-readable medium" as used herein refers to any medium
that participates in providing instructions to a processor for execution.
Such a medium may take many forms, including but not limited to
non-volatile media, volatile media, and transmission media. Non-volatile
media may include, for example, optical or magnetic disks. Volatile media
may include dynamic memory. Transmission media may include coaxial
cables, copper wire and fiber optics. Transmission media may also take
the form of acoustic, optical, or electromagnetic waves, such as those
generated during radio frequency (RF) and infrared (IR) data
communications. Common forms of computer-readable media include, for
example, a floppy disk, a flexible disk, hard disk, magnetic tape, or
other magnetic medium, a CD-ROM, CDRW, DVD, or other optical medium,
punch cards, paper tape, optical mark sheets, or other physical medium
with patterns of holes or other optically recognizable indicia, a RAM, a
PROM, and EPROM, a FLASH-EPROM, or other memory chip or cartridge, a
carrier wave, or other medium from which a computer can read.

[0066]A particular profile may be coupled with additional data about that
profile on a computer readable medium. For instance, a profile may be
coupled with data about what therapeutics, compounds, or drugs may be
efficacious for that profile. Conversely, a profile may be coupled with
data about what therapeutics, compounds, or drugs may not be efficacious
for that profile. Alternatively, a profile may be coupled with known
risks associated with that profile. Non-limiting examples of the type of
risks that might be coupled with a profile include disease or disorder
risks associated with a profile. The computer readable medium may also
comprise a database of at least two distinct profiles.

[0067]Profiles may be stored on a computer-readable medium such that
software known in the art and detailed in the examples may be used to
compare more than one profile.

[0068]Another aspect of the invention is a method for selecting a compound
that has efficacy for modulating a gene product of M. smithii present in
the gastrointestinal tract of a subject. The method generally comprises
comparing an M. smithii gene profile to a gene profile of the subject and
identifying a gene product of the M. smithii gene profile that is
divergent from a corresponding gene product of the subject gene profile,
or absent in the gene profile of the subject. Next the method comprises
selecting a compound that modulates the M. smithii gene product, but does
not substantially modulate the corresponding gene product of the subject.
In a further embodiment, the compound also does not substantially
modulate the corresponding gene product of an archaeon other than M.
smithii, or a non-archaeal microbe, in the gastrointestinal tract of the
subject. The compound may for instance, inhibit or promote the growth of
M. smithii. The compound may also decrease or increase the efficiency of
carbohydrate metabolism in the subject. Accordingly, the compound may
also promote weight loss or weight gain in the subject.

[0069]Another further aspect of the invention is a method for selecting a
compound that has efficacy for modulating a gene product of M. smithii
present in the gastrointestinal tract of a subject. The method comprises
comparing an M. smithii gene profile to a gene profile of the subject and
identifying a gene product of the M. smithii gene profile that is
divergent from a corresponding gene product of the subject gene profile,
or absent in the gene profile of the subject. Next the method comprises
selecting a compound that can be administered so as to modulate the M.
smithii gene product, but not substantially modulate the corresponding
gene product of the subject. In a further embodiment, the administered
compound also does not substantially modulate the corresponding gene
product of an archaeon other than M. smithii, or a non-archaeal microbe,
in the gastrointestinal tract of the subject. The compound may be
administered, for instance, so as to inhibit or promote the growth of M.
smithii. The compound may also be administered so as to decrease or
increase the efficiency of carbohydrate metabolism in the subject.
Accordingly, the compound may also be administered so as to promote
weight loss or weight gain in the subject.

[0070]The present invention also encompasses a kit for evaluating a
compound, therapeutic, or drug. Typically, the kit comprises an array and
a computer-readable medium. The array may comprise a substrate having
disposed thereon at least one biomolecule that is derived from the M.
smithii genome or proteome. In some embodiments, the array may comprise
at least one biomolecule that is derived from the M. smithii metabolome
or transcriptome. The computer-readable medium may have a plurality of
digitally-encoded profiles wherein each profile of the plurality has a
plurality of values, each value representing the abundance of a
biomolecule derived from M. smithii detected by the array. The array may
be used to determine a profile for a particular subject under particular
conditions, and then the computer-readable medium may be used to
determine if the profile is similar to known profile stored on the
computer-readable medium. Non-limiting examples of possible known
profiles include obese and lean profiles for several different subjects.

III. Method of Promoting Weight Loss or Gain

[0071]A further aspect of the invention encompasses a method of promoting
weight loss or gain. The method incorporates the discovery that
modulating the Archaeon population of the gastrointestinal tract of a
subject, of which M. smithii is a major component, modulates the
efficiency and selectivity of carbohydrate metabolism. Furthermore, the
method relies on applicants' discovery that certain M. smithii gene
products are conserved among M. smithii strains, yet divergent (or
absent) from the correlating gene products expressed by the subject's
microbiome or genome. This divergence allows the selection of compounds
to specifically modulate the M. smithii gene product, while substantially
not modulating the subject's gene product, as described above.

[0072]By way of non-limiting example, weight loss may be promoted by
administering an HMG-CoA reductase inhibitor to a subject. In an
exemplary embodiment, the inhibitor will selectively inhibit the HMG-CoA
reductase expressed by M. smithii and not the HMG-CoA reductase expressed
by the subject. In another embodiment, a second HMG CoA-reductase
inhibitor may be administered that selectively inhibits the HMG
CoA-reductase expressed by the subject in lieu of the HMG-CoA reductase
expressed by M. smithii. In yet another embodiment, an HMG-CoA reductase
inhibitor that selectively inhibits the HMG-CoA reductase expressed by
the subject may be administered in combination with an HMG-CoA reductase
inhibitor that selectively inhibits the HMG-CoA reductase expressed by M.
smithii. One means that may be utilized to achieve such selectivity is
via the use of time-release formulations as discussed below. Compounds
that inhibit HMG-CoA reductase are well known in the art. For instance,
non-limiting examples include atorvastatin, pravastatin, rosuvastatin,
and other statins.

(a) Pharmaceutical Compositions

[0073]These compounds, for example HMG-CoA reductase inhibitors, may be
formulated into pharmaceutical compositions and administered to subjects
to promote weight loss. According to the present invention, a
pharmaceutical composition includes, but is not limited to,
pharmaceutically acceptable salts, esters, salts of such esters, or any
other adduct or derivative which upon administration to a subject in need
is capable of providing, directly or indirectly, a composition as
otherwise described herein, or a metabolite or residue thereof, e.g., a
prodrug.

[0074]The pharmaceutical compositions maybe administered by several
different means that will deliver a therapeutically effective dose. Such
compositions can be administered orally, parenterally, by inhalation
spray, rectally, intradermally, intracisternally, intraperitoneally,
transdermally, bucally, as an oral or nasal spray, or topically (i.e.
powders, ointments or drops) in dosage unit formulations containing
conventional nontoxic pharmaceutically acceptable carriers, adjuvants,
and vehicles as desired. Topical administration may also involve the use
of transdermal administration such as transdermal patches or
iontophoresis devices. The term parenteral as used herein includes
subcutaneous, intravenous, intramuscular, or intrasternal injection, or
infusion techniques. In an exemplary embodiment, the pharmaceutical
composition will be administered in an oral dosage form. Formulation of
drugs is discussed in, for example, Hoover, John E., Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and
Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,
Marcel Decker, New York, N.Y. (1980).

[0075]The amount of an HMG-CoA reductase inhibitor that constitutes an
"effective amount" can and will vary. The amount will depend upon a
variety of factors, including whether the administration is in single or
multiple doses, and individual subject parameters including age, physical
condition, size, and weight. Those skilled in the art will appreciate
that dosages may also be determined with guidance from Goodman &
Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition
(1996), Appendix II, pp. 1707-1711 and from Goodman & Goldman's The
Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II,
pp. 475-493.

(b) Controlled Release Formulations

[0076]As described above, an HMG-CoA reductase inhibitor may be specific
for the M. smithii enzyme, or for the subject's enzyme, depending, in
part, on the selectivity of the particular inhibitor and the area the
inhibitor is targeted for release in the subject. For example, an
inhibitor may be targeted for release in the upper portion of the
gastrointestinal tract of a subject to substantially inhibit the
subject's enzyme. In contrast, the inhibitor may be targeted for release
in the lower portion of the gastrointestinal tract of a subject, i.e.,
where M. smithii resides, then the inhibitor may substantially inhibit M.
smithii's enzyme.

[0077]In order to selectively control the release of an inhibitor to a
particular region of the gastrointestinal tract for release, the
pharmaceutical compositions of the invention may be manufactured into one
or several dosage forms for the controlled, sustained or timed release of
one or more of the ingredients. In this context, typically one or more of
the ingredients forming the pharmaceutical composition is
microencapsulated or dry coated prior to being formulated into one of the
above forms. By varying the amount and type of coating and its thickness,
the timing and location of release of a given ingredient or several
ingredients (in either the same dosage form, such as a multi-layered
capsule, or different dosage forms) may be varied.

[0078]The coating can and will vary depending upon a variety of factors,
including, the particular ingredient, and the purpose to be achieved by
its encapsulation (e.g., time release). The coating material may be a
biopolymer, a semi-synthetic polymer, or a mixture thereof. The
microcapsule may comprise one coating layer or many coating layers, of
which the layers may be of the same material or different materials. In
one embodiment, the coating material may comprise a polysaccharide or a
mixture of saccharides and glycoproteins extracted from a plant, fungus,
or microbe. Non-limiting examples include corn starch, wheat starch,
potato starch, tapioca starch, cellulose, hemicellulose, dextrans,
maltodextrin, cyclodextrins, inulins, pectin, mannans, gum arabic, locust
bean gum, mesquite gum, guar gum, gum karaya, gum ghatti, tragacanth gum,
funori, carrageenans, agar, alginates, chitosans, or gellan gum. In
another embodiment, the coating material may comprise a protein. Suitable
proteins include, but are not limited to, gelatin, casein, collagen, whey
proteins, soy proteins, rice protein, and corn proteins. In an alternate
embodiment, the coating material may comprise a fat or oil, and in
particular, a high temperature melting fat or oil. The fat or oil may be
hydrogenated or partially hydrogenated, and preferably is derived from a
plant. The fat or oil may comprise glycerides, free fatty acids, fatty
acid esters, or a mixture thereof. In still another embodiment, the
coating material may comprise an edible wax. Edible waxes may be derived
from animals, insects, or plants. Non-limiting examples include beeswax,
lanolin, bayberry wax, carnauba wax, and rice bran wax. The coating
material may also comprise a mixture of biopolymers. As an example, the
coating material may comprise a mixture of a polysaccharide and a fat.

[0079]In an exemplary embodiment, the coating may be an enteric coating.
The enteric coating generally will provide for controlled release of the
ingredient, such that drug release can be accomplished at some generally
predictable location in the lower intestinal tract below the point at
which drug release would occur without the enteric coating. In certain
embodiments, multiple enteric coatings may be utilized. Multiple enteric
coatings, in certain embodiments, may be selected to release the
ingredient or combination of ingredients at various regions in the lower
gastrointestinal tract and at various times.

[0080]The enteric coating is typically, although not necessarily, a
polymeric material that is pH sensitive. A variety of anionic polymers
exhibiting a pH-dependent solubility profile may be suitably used as an
enteric coating in the practice of the present invention to achieve
delivery of the active to the lower gastrointestinal tract. Suitable
enteric coating materials include, but are not limited to: cellulosic
polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose,
hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose,
cellulose acetate, cellulose acetate phthalate, cellulose acetate
trimellitate, hydroxypropylmethyl cellulose phthalate,
hydroxypropylmethyl cellulose succinate and carboxymethylcellulose
sodium; acrylic acid polymers and copolymers, preferably formed from
acrylic acid, methacrylic acid, methyl acrylate, ammonio methylacrylate,
ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (e.g.,
those copolymers sold under the trade name "Eudragit"); vinyl polymers
and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate,
polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and
ethylene-vinyl acetate copolymers; and shellac (purified lac). In one
embodiment, the coating may comprise plant polysaccharides that can only
be digested in the distal gut by the microbiota. For instance, a coating
may comprise pectic galactans, polygalacturonates, arabinogalactans,
arabinans, or rhamnogalacturonans. Combinations of different coating
materials may also be used to coat a single capsule.

[0081]The thickness of a microcapsule coating may be an important factor
in some instances. For example, the "coating weight," or relative amount
of coating material per dosage form, generally dictates the time interval
between oral ingestion and drug release. As such, a coating utilized for
time release of the ingredient or combination of ingredients into the
gastrointestinal tract is typically applied to a sufficient thickness
such that the entire coating does not dissolve in the gastrointestinal
fluids at pH below about 5, but does dissolve at pH about 5 and above.
The thickness of the coating is generally optimized to achieve release of
the ingredient at approximately the desired time and location.

[0082]As will be appreciated by a skilled artisan, the encapsulation or
coating method can and will vary depending upon the ingredients used to
form the pharmaceutical composition and coating, and the desired physical
characteristics of the microcapsules themselves. Additionally, more than
one encapsulation method may be employed so as to create a multi-layered
microcapsule, or the same encapsulation method may be employed
sequentially so as to create a multi-layered microcapsule. Suitable
methods of microencapsulation may include spray drying, spinning disk
encapsulation (also known as rotational suspension separation
encapsulation), supercritical fluid encapsulation, air suspension
microencapsulation, fluidized bed encapsulation, spray cooling/chilling
(including matrix encapsulation), extrusion encapsulation, centrifugal
extrusion, coacervation, alginate beads, liposome encapsulation,
inclusion encapsulation, colloidosome encapsulation, sol-gel
microencapsulation, and other methods of microencapsulation known in the
art. Detailed information concerning materials, equipment and processes
for preparing coated dosage forms may be found in Pharmaceutical Dosage
Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc.,
1989), and in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery
Systems, 6th Ed. (Media, Pa.: Williams & Wilkins, 1995).

DEFINITIONS

[0083]The term "activity of the microbiota population" refers to the
microbiome's ability to harvest energy.

[0084]An "effective amount" is a therapeutically-effective amount that is
intended to qualify the amount of agent that will achieve the goal of
modulating an M. smithii gene product, promoting weight loss, or
promoting weight gain.

[0085]As used herein, "gene product" refers to a nucleic acid derived from
a particular gene, or a polypeptide derived from a particular gene. For
instance, a gene product may be a mRNA, tRNA, rRNA, cDNA, peptide,
polypeptide, protein, or metabolite.

[0086]"Metabolome" as used herein is defined as the network of enzymes and
their substrates and biochemical products, which operate within subject
or microbial cells under various physiological conditions.

[0087]As used herein, the term "pharmaceutically acceptable salt" refers
to those salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and other subjects
without undue toxicity, irritation, allergic response and the like, and
are commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well known in the art. For example, S. M. Berge, et
al. describe pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences, 66: 1 19 (1977), incorporated herein by
reference. The salts can be prepared in situ during the final isolation
and purification of the composition of the invention, or separately by
reacting the free base function with a suitable organic acid.
Non-limiting examples of pharmaceutically acceptable, nontoxic acid
addition salts are salts of an amino group formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, hydroionic acid, nitric
acid, carbonic acid, phosphoric acid, sulfuric acid and perchloric acid.

[0088]As used herein, the "subject" may be, generally speaking, an
organism capable of supporting M. smithii in its gastrointestinal tract.
For instance, the subject may be a rodent or a human. In one embodiment,
the subject may be a rodent, i.e. a mouse, a rat, a guinea pig, etc. In
an exemplary embodiment, the subject is human.

[0089]"Transcriptome" as used herein is defined as the network of genes
that are being actively transcribed into mRNA in subject or microbial
cells under various physiological conditions.

[0091]As various changes could be made in the above compounds, products
and methods without departing from the scope of the invention, it is
intended that all matter contained in the above description and in the
examples given below, shall be interpreted as illustrative and not in a
limiting sense.

EXAMPLES

[0092]The following examples illustrate various iterations of the
invention.

Materials and Methods for the Examples

Genome Sequencing and Annotation

[0093]Methanobrevibacter smithii strain PS (ATCC 35061) was grown as
described below for 6 d at 37° C. DNA was recovered from harvested
cell pellets using the QIAGEN Genomic DNA Isolation kit with mutanolysin
(1 unit/mg wet weight cell pellet; Sigma) added to facilitate lysis of
the microbe. An ABI 3730xl instrument was used for paired end-sequencing
of inserts in a plasmid library (average insert size 5 Kb; 42,823 reads;
11.6×-fold coverage), and a fosmid library (average insert size of
40 Kb; 7,913 reads; 0.6×-fold coverage). Phrap and PCAP (Huang et
al. (2003) Genome Res 13:2164-70) were used to assemble the reads. A
primer-walking approach was used to fill-in sequence gaps. Physical gaps
and regions of poor quality (as defined by Consed; Gordon et al., (1998)
Genome Res. 8, 195-202) were resolved by PCR-based re-sequencing. The
assembly's integrity and accuracy was verified by clone constraints.
Regions containing insufficient coverage or ambiguous assemblies were
resolved by sequencing spanning fosmids. Sequence inversions were
identified based on inconsistency of constraints for a fraction of read
pairs in those regions. The final assembly consisted of 12.6×
sequence coverage with a Phred base quality value 40. Open-reading frames
(ORFs) were identified and annotated as described below.

[0096]M. smithii PS was also cultured in a BioFlor-110 batch fermentor
with dual 1.5 L fermentation vessels (New Brunswick Scientific). Each
vessel contained 750 ml of supplemented MBC medium. One hour prior to
inoculation, 7.5 ml of sterile 2.5% Na2S solution was added to the
vessel, followed by one half of the contents of a serum bottle culture
that had been harvested on day 5 of growth. Microbes were then incubated
at 37° C. under a constant flow of H2/CO2 (4:1)
(agitation setting, 250 rpm). One milliliter of a sterile solution of
2.5% Na2S was added daily.

Colonization of Germ-Free Mice with M. smithii PS with and without B.
thetaiotaomicron VPI-5482

[0097]Mice belonging to the NMRI/KI inbred strain (Bry et al., (1996)
Science 273:1380-3) were housed in gnotobiotic isolators (Hooper et al.,
(2002) Mol Cell Micro 31:559-589) where they were maintained under a
strict 12 h light cycle (lights on at 0600 h) and fed a standard,
autoclaved, polysaccharide-rich chow diet (B&K Universal, East Yorkshire,
UK) ad libitum. Each mouse was inoculated at age 8 weeks with a single
gavage of 108 microbes/strain [B. thetaiotaomicron was harvested
from an overnight culture in TYG medium (Sonnenburg et al., Science
307:1955-9); M. smithii from serum bottles containing MBC medium after a
5 d incubation at 37° C. (Samuel et al., (2006) PNAS
103:10011-6)]. For a given experiment, the same preparation of cultured
microbes was used for mono-association (single species added) and
co-colonization (both species added).

[0100]GO term assignments--The number of genes in each archaeal genome
that were assigned to each GO term, or to its parents in the GO hierarchy
[version available on Jun. 6, 2006; (Ashburner et al., (2000) Nat Genet
25:25-9)] were totaled. All terms assigned to at least five genes in a
given genome were then subjected to statistical tests for
overrepresentation, and all terms with a total of five genes across all
tested genomes for under-representation, using a binomial comparison
reference set (see Table 6). Genes that could not be assigned to a GO
category were excluded from the reference sets. A false discovery rate of
<0.05 was set for each comparison (Benjamini et al., (1995) J of the
Royal Statistical Society B 57:289-300). All tests were implemented using
the Math::CDF Perl module (E. Callahan, Environmental Statistics,
Fountain City, Wis.; available at http://www.cpan.org/), and scripts
written in Perl.

[0102]Genomic synteny--Comparisons of synteny between M. smithii and M.
stadtmanae were completed using the Artemis Comparison Tool (Carver et
al., (2005) Bioinformatics 21:3422-3) set to tBLASTX and the most
stringent confidence level.

[0103]M. smithii interaction network analyses--All M. smithii COGs were
submitted to the STRING database (http://string.embl.de/; (von Mering et
al., (2003) Nucleic Acids Res 31:258-61) to create predicted interaction
networks (0.95 confidence interval). The program Medusa (Hooper et al.,
(2005) Bioinformatics 21:4432-3) was then used to organize the networks
and color the nodes based on their conservation in M. smithii's proteome
(mutual best BLASTP hits with e-values <10-20 to the other
Methanobacteriales genomes).

[0104]Clustering of adhesin-like proteins--M. smithii and M. stadtmanae
ALPs were first aligned using CLUSTALW (v.1.83; (Chenna et al., (2003)
Nucleic Acids Res 31:3497-500)). To retain the highest level of
discrimination between the proteins, the alignment was subsequently
converted into a nucleotide alignment using PAL2NAL (Suyama et al.,
(2006) Nucleic Acids Res 34:W609-12). The resulting alignment was used to
create a maximum likelihood tree with RAxML [Randomized accelerated
maximum likelihood for high performance computing [RAxML-VI-HPC, v2.2.1;
(Stamatakis (2006) Bioinformatics 22:2688-90)] first using the GTR+CAT
approximation method for rapid generation of tree topology, followed by
the GTR+gamma evolutionary model for determination of likelihood values.
ModelTest (v3.7; http://darwin.uvigo.es/software/modeltest.html) also
identified GTR+gamma as the most appropriate evolutionary model for the
dataset. Bootstrap values were determined from 100 neighbor-joining trees
in Paup (v. 4.0b10, http://paup.csit.fsu.edu/). Tree visualization was
completed with TreeView (Page (1996) Comput Appl Biosci 12:357-8).

[0108]Cells were harvested at day 6 of growth in the batch fermentor, and
cellular morphology was defined by TEM using methods identical to those
described previously for B. thetaiotaomicron (Sonnenburg et al., (2005)
Science 307:1955-9). TEM studies of M. smithii present in the ceca of
gnotobiotic mice that had been colonized for 14 d with the archaeon were
conducted using the same protocol.

[0109]Extraction of metabolites from cecal contents--For measurement of
ammonia and urea levels, perchloric acid extracts were prepared from 2 mg
of freeze-dried cecal contents. [Contents were collected with a 10 μl
inoculation loop, quick frozen in liquid nitrogen, and lyophilized at
-35° C.] The lyophilized sample was homogenized in 0.2 ml of 0.3M
perchloric acid at 1° C.

[0110]For the remaining metabolites, alkali and acid extracts were
prepared from 4 mg of dried cecal samples that were homogenized in 0.4 ml
0.2M NaOH at 1° C. For the alkali extract, an 80 μl aliquot was
removed, heated for 20 min at 80° C. and then neutralized with 80
μl of 0.25M HCl and 100 mM Tris base. For the acid extract, a 60 μl
aliquot was removed and added to 20 μl 0.7M HCl, heated for 20 min at
80° C., and then neutralized with 40 μl 100 mM Tris base.
Protein content was determined in the alkali extracts using the Bradford
method (Bio Rad).

[0111]Metabolite assays--The sample concentrations for ammonium and urea
were high enough so that direct fluorometric measurements could be used
for detection. However, to measure the low sample concentrations for
asparagine, glutamate, glutamine, α-ketoglutarate and ethanol,
protocols were adapted from previously established pyridine
nucleotide-linked assays, an "oil well" technique, and enzymatic cycling
amplification (Passonneau et al., (1993) Enzymatic Analysis:A Practical
Guide). All chemicals and enzymes were from Sigma unless otherwise noted.

[0113]To measure urea concentrations, 2 μl of a 50 mg/ml solution of
Jack bean urease (50 units/mg) was added to the same sample used to
determine ammonium levels. Following a 40 min incubation at 24°
C., urea levels were defined based on a further reduction in
fluorescence. Control sample blanks lacked added urease. Reference urea
standards were carried throughout all steps.

[0117]Ethanol: A 0.5 μl aliquot of an acid extract from cecal contents
was added to 0.5 μl of a solution consisting of 5 mM Tris HCl (pH
8.1), 0.04% BSA, 0.1 mM NAD+, and 20 μg/ml yeast alcohol dehydrogenase
(350 units/mg protein). Following a 60 min incubation at 24° C., 1
μl of 0.15M NaOH was added and the mixture heated for 20 min at
80° C. A 0.5 μl aliquot of this reaction mixture was
transferred to 0.1 ml of NAD cycling reagent and amplified 5000-fold.
Ethanol standards were carried throughout all steps.

Whole Genome Genotyping with Custom M. smithii GeneChips

[0118]GeneChips were manufactured by Affymetrix
(http://www.affymetrix.com), based on the sequence of the PS strain
genome (see Table 12 for details of the GeneChip design). Duplicate
cultures of M. smithii strains PS (ATCC 35061), F1 (DSMZ 2374), ALI (DSMZ
2375) and B181 (DSMZ 11975), were grown in 125 ml serum bottles as
described above. Genomic DNA was prepared from each strain using the
QIAGEN Genomic DNA Isolation kit: mutanolysin (Sigma; 2.5 U/mg wet wt.
cell pellet) was added to facilitate lysis of the microbes. DNA (5-7
μg) was further purified by phenolchloroform extraction and then
sheared by sonication to <200 bp, labeled with biotin (Enzo BioArray
Terminal Labeling Kit), denatured at 95° C. for 5 min, and
hybridized to replicate GeneChips using standard Affymetrix protocols
(http://www.affymetrix.com). M. smithii genes represented on the GeneChip
were called "Present" or "Absent" by DNA-Chip Analyzer v1.3 (dChip;
www.biostat.harvard.edu/complab/dchip/) using modeled (PM/MM ratio) data.

Statistical Analysis

[0119]Pairwise comparisons were made using unpaired Student's t-test.
One-way ANOVA, followed by Tukey's post hoc multiple comparison test, was
used to determine the statistical significance of differences observed
between three groups.

Development of PHAT (Pressurized Heated Anaerobic Tank) System

[0120]A system for culturing M. smithii in 96-well plate format was
designed and constructed in the following manner (See FIG. 15). Three
stainless steel paint canisters (Binks, 83S-210, 2 gallon size) were
modified for incubation of plates at 37° C. in an oxygen-free gas
mix of 20% CO2/80% H2 at a pressure of 30 psi, where all of
these growth parameters can be monitored and recorded.

[0121]The canisters are heated using Electro-Flex Heat brand Pail Heaters
controlled by a custom designed controller consisting of a 16A2120
temperature/process control (Love Controls), an RTD (resistance
temperature detector) probe to measure internal tank temperature, and
several safety features to prevent overheating or burns.

[0122]The system is pressurized with oxygen-free gas that has flowed
through a custom-built oxygen scrub. Commercially available gas mixes
used for culturing M. smithii contain trace levels of oxygen that would
kill the organism: thus, the gas mixture must be passed through an oxygen
scrub. This scrub consists of a glass tube filled with copper mesh that
is heated to 350° C. with heating tape (HTS/Amptek Duo-Tape),
controlled by a benchtop power controller (HTS/Amptek BT-Z). The oxygen
scrub is covered with insulating tape and secured behind a heat resistant
polyetherimide case. Pressure in each tank is measured and recorded with
a digital manometer (LEO record, Omni Instruments).

[0123]The system is housed inside an anaerobic chamber (COY laboratories)
to allow inspection and manipulation of cultures and plates without
exposing M. smithii to oxygen. Each tank can house 30 standard volume
96-well plates, which can be analyzed inside the COY anaerobic chamber
with a microplate reader (BioRad) that monitors growth by measuring
optical density.

Statin Susceptibility

[0124]Stock solutions (100×) of atorvastatin were prepared in
methanol, pravastatin in ethanol, and rosuvastatin in DMSO (dimethyl
sulfoxide) to concentrations of 100 mM, 10 mM and 1 mM. 1.5 μl of the
stock solutions were added to wells in 96-well plates and transferred to
the COY anaerobic chamber where they were kept for at least 24 hours to
become anaerobic. 150 microliters of actively growing Methanobrevibacter
smithii cultures were then added to each well (excluding medium+drug
blanks) to bring the drug concentrations to 1 mM, 100 μM and 10 μM,
respectively. The plates were incubated in the newly developed
pressurized heated anaerobic tank system in a 4:1 mixture of
oxygen-scrubbed H2 and CO2 at a pressure of 30 psi. Cultures
grown in 1% ethanol, methanol and DMSO were used as controls. Growth was
measured by determining optical density at 600 nm using the BioRad
microplate reader (model 680).

[0125]Starting cultures of M. smithii strains [DSMZ 861 (PS), 2374 (F1),
2375 (ALI) and 11975 (B181)] were grown in 96 well plates in 150 μl
volume/well of Methanobrevibacter complex medium (MBC) supplemented with
3 g/liter formate, 3 g/liter acetate, and 33 ml/liter of 2.5% Na2S
(added just before use). Each condition was tested in triplicate with the
average measurement plotted.

[0128]Several lytic phages have been reported to infect M. smithii,
including a 69 kb linear phage known as PG that belongs to the
ψM1-like viruses (Prangishvili et al. (2006) Virus Res 117:52-67),
and another 35 kb phage (PMS11; Calendar (2005) The Bacteriophages). The
PG phage is AT-rich, heavily nicked, and lytic (burst size, 30-90), with
a latent period of 3-4 h (Bertani et al. (1985) EMBO Workshop on
Molecular Genetics of Archaebacteria and the International Workshop on
Biology and Biochemistry of Archaebacteria, pg. 398). BLAST comparisons
of the 52 predicted genes in the integrated prophage of M. smithii PS
against known phage genes revealed only a few homologs (Table 13). One of
the prophage genes (MSM1691) encodes a pseudomurein endoisopeptidase
(PeiW): this enzyme may function to cleave M. smithii's cell wall and
contribute to autolysis, as related enzymes in a defective
Methanothermobacter wolfeii prophage have been shown to do (Luo et al.,
FEMS Microbiology Letters 208:47-51). The specific ends of the prophage
genome could not be identified, and further studies are needed to
determine whether the prophage is active and lytic.

[0130]M. smithii PS contains 60 predicted transcriptional regulators,
including homologs of known nutrient sensors [e.g., a HypF family member
(maturation of hydrogenases), a PhoU family member (phosphate
metabolism), and a NikR family member (nickel)], plus five regulators of
amino acid metabolism (Table 3). However, several GO categories related
to environmental sensing and regulation (e.g., two-component systems;
GO:0000160) are significantly depleted in its proteome compared to the
proteomes of methanogens that live in terrestrial or aquatic environments
(Table 6). In contrast, B. thetaiotaomicron, which uses complex,
structurally diversified glycans as its principal nutrient source,
possesses a large and diverse arsenal of nutrient sensors including 32
hybrid two-component systems plus 50 ECF-type sigma factors and 25
anti-sigma factors (Sonnenburg et al, (2006) PNAS 103:8834-9; Xu et al.,
(2003) Science 299:2074-6). This relative paucity of nutrient sensors may
reflect the fact that M. smithii's niche is restricted, and its nutrient
substrates are relatively small, readily diffusible molecules that may
not require extensive machinery for their recognition.

Bile Acid Detoxification

[0131]In humans, cholic and chenodeoxycholic acids are synthesized in the
liver and during their enterohepatic circulation undergo transformation
by the intestinal microbiota to an array of metabolites (Hylemon and
Harder (1998) FEMS Microbiol Rev 22:475-88). Bile acids and their
metabolites have microbicidal activity and a genetically engineered
deficiency of the bile acid-activated nuclear receptor FXR leads to
reduced bile acid pools and bacterial overgrowth (Inagaki et al., (2006)
PNAS 103:3920-5). Both M. smithii and M. stadtmanae encode a sodium:bile
acid symporter (MSM1078), a conjugated bile acid hydrolase (CBAH;
MSM0986), a short chain dehydrogenase with homology to a
7α-hydroxysteroid dehydrogenase (MSM0021). This is consistent with
in vitro studies of M. smithii that demonstrate it is not inhibited by
0.1% deoxycholic acid (Miller et al, (1982) Appl Environ Microbiol
43:227-32).

[0132]We compared the proteome of M. smithii with the proteomes of (i)
Methanosphaera stadtmanae, a methanogenic Euryarchaeote that is a minor
and inconsistent member of the human gut microbiota (Eckburg et al.,
(2005) Science 308:1635-38), (ii) nine `non-gut methanogens` recovered
from microbial communities in the environment, and (iii) these non-gut
methanogens plus an additional 17 sequenced Archaea (`all archaea`)
(Table 5).

[0135]The ability to vary capsular polysaccharide surface structures in
vivo by altering expression of glycosyltransferases (GTs) is a feature
shared among sequenced bacterial species that are prominent in the distal
human gut microbiota (Sonnenburg et al., (2005) Science 307:1955-59;
Sonnenburg et al., (2006) PNAS 103:8834-39; Mazmanian et al., (2005) Cell
122:107-118; Coyne et al., (2005) Science 307:1778-81). Transmission EM
studies of M. smithii harvested from gnotobiotic mice after a 14 day
colonization revealed that it too has a prominent capsule (FIG. 1A). The
proteomes of both human gut methanogens also contain an arsenal of GTs
[26 in M. smithii and 31 in M. stadtmanae; see Table 10 for a complete
list organized based on the Carbohydrate Active enZyme (CAZy)
classification scheme (http://www.cazy.org; (Coutinho et al., (1999)
Recent Advances in Carbohydrate Bioengineering)]. Unlike the sequenced
Bacteroidetes, which possess large repertoires of glycoside hydrolases
(GH) and carbohydrate esterases (CE) not represented in the human
`glycobiome`, neither gut methanogen has any detectable GH or CE family
members (FIG. 1B). Both M. smithii and M. stadtmanae dedicate a
significantly larger proportion of their `glycobiome` to GT2 family
glycosyltransferases than any of the sequenced nongut associated
methanogens (binomial test; p<0.00005; FIG. 1B). These GT2 family
enzymes have diverse predicted activities, including synthesis of
hyaluronan, a component of human glycosaminoglycans in the mucosal layer.

[0136]Sialic acids are a family of nine-carbon sugars that are abundantly
represented in human mucus- and epithelial cell surface-associated
glycans (Vimr et al., (2004) Microbiol Mol Biol Rev 68:132-53).
N-acetylneuraminic acid (Neu5Ac) is the predominant type of sialic acid
found in our species. Unique among sequenced archaea, M. smithii has a
cluster of genes (MSM1535-1540) that encode all enzymes necessary for de
novo synthesis of sialic acid from UDP-N-acetylglucosamine (i.e.
UDP-GlcNAc epimerase, Neu5Ac synthase, CMP-Neu5Ac synthetase, and a
putative polysialtransferase) (FIG. 1C). Biochemical analysis of extracts
prepared from cultured M. smithii, plus histochemical staining of the
microbe with the sialic-acid specific lectin, Sambucus nigra 1 agglutinin
(SNA), confirmed the presence of a molecular species that co-elutes with
a sialic acid standard in this analytic HPLC system (FIG. 6A-C). Taken
together, our findings indicate that M. smithii has developed mechanisms
to decorate its surface with carbohydrate moieties that mimic those
encountered in the glycan landscape of its intestinal habitat.

[0137]The genomes of both human gut methanogens also encode a novel class
of predicted surface proteins that have features similar to bacterial
adhesins (48 members in M. smithii and 37 in M. stadtmanae). A
phylogenetic analysis indicated that each methanogen has a specific clade
of these Adhesin-Like Proteins (ALPs; FIG. 7). A subset of the M. smithii
ALPs has homology to pectin esterases (GO:0030599): this GO family, which
is significantly enriched in this compared to other Archaea based on the
binomial test (p<0.0005; Table 6), is associated with binding of
chondroitin, a major component of mucosal glycosaminoglycans. Several
other M. smithii ALPs have domains predicted to bind other sugar moieties
(e.g. galactose-containing-glycans; FIG. 7A). Both methanogens also have
ALPs with peptidase-like domains (see Table 11 for a complete list of
InterPro domains).

Example 2

Methanogenic and Non-Methanogenic Removal of Bacterial End-Products of
Fermentation

[0138]Compared to other sequenced non-gut associated methanogens, M.
smithii has significant enrichment of genes involved in utilization of
CO2, H2 and formate for methanogenesis (GO:0015948; Table 6).
They include genes that encode proteins involved in synthesis of vitamin
cofactors used by enzymes in the methanogenesis pathway [methyl group
carriers (F430 and corrinoids); riboflavin (precursor for F430
biosynthesis); and coenzyme M synthase (involved in the terminal step of
methanogenesis)] (see Table 7 for a list of these genes, and FIG. 2A for
the metabolic pathways). M. smithii also has an intact pathway for
molybdopterin biosynthesis to allow for CO2 utilization (FIG. 8). M.
smithii also upregulates a formate utilization gene cluster (FdhCAB;
MSM1403-5) for methanogenic consumption of this B.
thetaiotaomicron-produced metabolite (Samuel and Gordon (2006) PNAS
103:10011-10016).

[0141]Collectively, these findings indicate that M. smithii supports
methanogenic and non-methanogenic removal of diverse bacterial
end-products of fermentation: this capacity may endow it with a great
flexibility to form syntrophic relationships with a broad range of
bacterial members of the distal human gut microbiota.

[0143]Microanalytic biochemical assays revealed a ratio of glutamine to
2-oxoglutarate concentration that was 15-fold lower in the ceca of
co-colonized gnotobiotic mice compared to animals colonized with M.
smithii alone, and 2-fold lower compared to B. thetaiotaomicron
mono-associated subjects (p<0.0001; FIG. 2C). In addition, levels of
several polar amino acids were also significantly reduced in mice with
the saccharolytic bacterium and methanogen (FIG. 2D), providing
additional evidence for a nitrogen-limited gut environment. The key M.
smithii genes involved in ammonia assimilation, particularly those in the
high affinity glutamine synthetase-glutamate synthase pathway are GlnA
(glutamine synthetase, MSM1418) and GltA/GltB (two subunits of glutamate
synthase, MSM0027, MSM0368); FIG. 2A. GeneChip analysis of the
transcriptional responses of B. thetaiotaomicron to co-colonization with
M. smithii indicated that it also upregulates a high affinity glutamine
synthase [BT4339; 2.4-fold vs. B. thetaiotaomicron monoassociated mice;
n=4-5 mice/group; p<0.001; (Samuel et al., (2006) PNAS
103:10011-10016)]. This prioritization of ammonium assimilation by B.
thetaiotaomicron and M. smithii is accompanied by a modest but not
statistically significant decrease in cecal ammonium levels in
co-colonized subjects (13.4±1.8 μmol/g dry weight of cecal contents
vs. 142.45±1.0 in M. smithii- and 14.4±0.9 in B.
thetaiotaomicron-monoassociated animals; n=5-15/group; FIG. 2E).
Together, these studies indicate that ammonium represents a source of
nitrogen for M. smithii when it exists in isolation in the gut of
gnotobiotic mice, and that it may compete with B. thetaiotaomicron for
this nutrient resource.

[0146]We designed a custom GeneChip containing probesets directed against
99.1% of M. smithii's 1795 known and predicted protein-coding genes (see
Table 12 for details). This GeneChip was used to perform whole genome
genotyping of M. smithii PS (control) plus three other strains recovered
from the feces of healthy humans: F1 (DSMZ 2374), ALI (DSMZ 2375) and
B181 (DSMZ 11975). Replicate hybridizations indicated that 100% of the
open reading frames (ORFs) represented on the GeneChip were detected in
M. smithii PS, while 90-94% were detected in the other strains, including
the potential drug targets mentioned above (Table 2 and FIG. 3).
Approximately 50% of the undetectable ORFs in each strain encode
hypothetical proteins. The other undetectable genes are involved in
genome evolution [e.g., recombinases, transposases, IS elements, and type
II restriction modification (R-M) systems], or are components of a
putative archaeal prophage in strain PS, or are related to surface
variation, including several ALPs (e.g., MSM0057 and MSM1585-90; FIG. 7).
Strains F1 and ALI also appear to lack redundant gene clusters encoding
subunits of formate dehydrogenase (MSM1462-3) and methyl-CoM reductase
(MSM0902-3) that are found in the PS strain (the latter cluster is also
undetectable in strain B181). In addition, the only methanol utilization
cluster present in the PS strain (MSM1515-8) was not detectable in strain
F1 (Table 2).

[0147]To further assess the degree of nucleotide sequence divergence among
M. smithii strains, we compared the sequenced PS type strain to a 78 Mb
metagenomic dataset generated from the aggregate fecal microbial
community genome (microbiome) of two healthy humans (Gill et al., (2006)
Science 312:1355-59). Their sequenced microbiomes contained 92% of the
ORFs in the type strain (Table 2), including the potential drug targets
described above. Several R-M system gene clusters (MSM0157-8, MSM1743,
MSM1746-7), a number of transposases, a DNA repair gene cluster
(MSM0689-95), and all ORFs in the prophage were not evident in the two
microbiomes. Sequence divergence was also observed in 33 of the 48 ALP
genes plus two `surface variation` gene clusters (MSM1289-1398 and
MSM1590-1616) that encode 11 glycosyltransferases and 9 proteins involved
in pseudomurein cell wall biosynthesis (FIG. 9). A redundant methyl-CoM
reductase cluster (MSM0902-3), an F420-dependent NADP oxidoreductase
(MSM0049) involved in consumption of bacteria-derived ethanol, and two
subunits of the bicarbonate ABC transporter (MSM0990-1; carbon
utilization) exhibited heterogeneity in the M. smithii populations
present in the gut microbiota of these two adults (Table 2 and FIG. 9).

[0148]In yet another type of analysis, we compared the sequenced genome of
M. smithii strain PS to the sequenced genomes of 11 other strains,
isolated from the fecal microbiota of a pair of adult female monozygotic
twins and two other unrelated individuals. The results, summarized in
Table A, reveal a set of 1436 genes that are represented in all of these
human isolates as well as the PS type strain. These genes, which include
the gene encoding HMG-CoA reductase, comprise a human gut-associated M.
smithii "core" genome.

Example 5

Effect of HMG-CoA Reductase Inhibitors Administration

[0149]The PHAT system was used to culture 4 strains of M. smithii (DSMZ
861 (PS), 2374 (F1), 2375 (ALI) and 11975 (B181)) in 96-well plate
format, and to test their sensitivities to various HMG-CoA reductase
inhibitors. Preliminary results indicate that atorvastatin
(Lipitor®), pravastatin (Pravachol®) and rosuvastatin
(Crestor®) inhibit all strains tested at concentrations of 1
millimolar. Atorvastatin and rosuvastatin also inhibit all strains at 100
micromolar concentrations (FIG. 10-13; Tables 14-17). None of these three
statins had any affect on the growth of a dominant human gut-associated
saccharolytic bacterium, Bacteroides thetaiotaomicron (FIG. 14).

Methods). Note that the term `absent`is
based on different criteria than those used for the human microbiome
dataset (see footnote 2).
2Metagenomic datasets from the microbiomes of two healthy lean adults
(Gill et al., 2006)
were tested for identity to M. smithii PS ORFs; ORFs with reads that
matched with >95% identity
are called `present`, 80-95% identity are called `divergent`, and <80%
identity are called `absent`.
iiProbeset for M. smithii gene not represented on GeneChip.